MCB 201 Gene Expression - Spring Semester 2003
Lecture 18 (Regulation of Transcription Initiation cont.)
Section 10.7 continued (Molecular Mechanisms of Eukaryotic Transcriptional Control)
1. Figure 10-59: A method for analyzing the acetylation state of histones in chromatin associated with a specific region of the genome. Note the use of a low level of reversible crosslinking of nucleosomes to DNA to start off. After shearing the chromatin to a small size, specific antibodies are used to immunoprecipitate certain fragments, in this case those with acetylated histones. The immunoprecipitates are further characterized by reversing the cross-linking, removing the protein, then denaturing the DNA and assaying it by polymerase chain reaction (PCR). By this method, one can determine which DNA sequences are in regions of chromatin with acetylated histones.
2. Chromatin-remodeling factors and activation. Regions of actively transcribed chromatin are known to be more susceptible to DNase digestion than untranscribed regions. However, some of the DNA even in actively transcribed regions is protected by nucleosomes. Chromatin-remodeling complexes have the ability to loosen up the interaction between DNA and nucleosomes even more, at least in part through their helicase activity. These regions then become hypersensitive to DNase activity.
3. How do transcriptional activators stimulate the assembly of an initiation complex and regulate the frequency at which new PolII polymerases start? They do this by interacting with general transcription factors and with the Mediator complex which binds to the CTD of PolII. It is these types of interactions that are responsible for the strong cooperativity in assembly of the initiation complex. This cooperative binding effect is a contributor to cell type-specific gene expression as well.
4. Figure 10-60: The regulation of the TTR gene in mammals, which encodes transthyretin is an example. This gene is expressed in hepatocytes where it is controlled by five different transcriptional activators. HNF = hepatocyte nuclear factor. Three of these activators are expressed in other cell types (AP1, C/EBP, HNF4), but transcription of the TTR gene occurs only in hepatocytes, where all five of these transcription factors are present. Different promoter regions that control hepatocyte-specific genes have different arrangements of binding sites and use overlapping but nonidentical sets of transcription factors, so there is no single unique hepatocyte-specific regulatory region.
5. Figure 10-61 (note media connection, Combinatorial Control of Transcription): All five of these transcription factors contribute to the cooperative assembly of an initiation complex at the TTR promoter in hepatocytes. Note the various genetic elements that play a role: enhancer, promoter-proximal region, TATA box, TTR gene coding sequence. Underneath are the proteins that bind to these regions. The activation domains of the bound activators (four of which are enriched in hepatocytes and AP1 which is broadly distributed in different mammalian cell types) have the possibility of interacting with any of the other components, resulting in the looping of the DNA and assembly of a stable, activated initiation complex. The activators have DNA binding sites in the enhancer region and in the promoter proximal region. Recall that the components shown to the right of the downpointing arrow are thought to be preassembled into a holoenzyme complex in vivo.
6. What is a repressor? Any protein that interferes with transcription initiation when it is bound to a specific site on DNA.
7. Figure 10-62: Three mechanisms by which the repressor may either inhibit or interfere with formation of an initiation complex. In addition to the mechanisms shown, some repressors bind to co-repressor proteins, which bind in turn to general transcription factors to inhibit initiation.
8. How are the transcription factors themselves controlled? The genes encoding these proteins are regulated at the level of transcription. In addition, because transcription factors can often exist in cells in a stored or inactive form, binding small effector molecules to them, such as lipid-soluble hormones like steroid hormones, and modifying them posttranslationally, by phosphorylation for example, can modulate their activities.
9. Let's look at the nuclear receptor superfamily of transcription factors as examples:
Figure 10-63: Examples of the ligands that bind to these TFs. All of these are lipid-soluble hormones that can pass through the plasma membrane of cells and enter the cytoplasm and nucleus.
Figure 10-64: The three domains shared by members of this superfamily and their level of evolutionary conservation. The variable region is the transcription activation domain, the DNA binding domain has a C4 zinc-finger motif, and the hormone binding domain is near the C-terminus.
Figure 10-65: The DNA binding sites for these TFs are called nuclear receptor response elements. Whether or not these short, repeated sequences are inverted or not has major significance for how the TFs bind. The TFs that bind to the inverted repeats are usually homodimers with two-fold axes of symmetry as shown previously in Figure 10-41b. The repeats that are oriented in the same direction are bound by heterodimeric TFs. These two classes of TFs differ in other respects as well.
10. Figure 10-67: An example of a homodimeric receptor is the glucocorticoid receptor. These types of receptors are located in both the nucleus and the cytoplasm, where they are held in an inactive complex with molecular chaperones, ready to interact with their ligands when they appear in the cell. When the steroid hormone ligand binds to the ligand binding domain (LBD), a major conformational change occurs in the receptor that causes it to dissociate from the molecular chaperone Hsp90 (There is a mistake on page 394 of Lodish; Hsp90 is not a protein related to Hsp70 as stated. These are unrelated proteins, but there is a protein in these complexes called Hsc70 which is related to stress-inducible Hsp70). The receptor can then move into the nucleus and bind to its response element.
11. Figure 10-66: Here is summarized an interesting experiment that demonstrates that the hormone-binding domain of the glucocorticoid receptor mediates translocation to the nucleus when hormone ligand is bound. Beta-galactosidase is used as a target for a fluorescent antibody as a way of determining the location of the glucocorticoid receptor and the effect of steroid hormone on its distribution. The top three panels show cells without hormone-treatment, i.e. they are controls. The bottom three panels show cells treated with dexamethasone, an artificial steroid, i.e. they are the so-called experimental cultures.
Panel A shows expression of only beta-galactosidase and no effect of hormone on its location as expected, another control; Panel B shows cells expressing the entire GR receptor attached to beta-galactosidase and showing nuclear localization in the presence of Dex; Panel C shows cells expressing beta-galactosidase attached to only the hormone-binding domain, again showing Dex-dependent nuclear localization.
12. Figure 10-68: Model of interferon gamma (IFNg) mediated gene activation by phosphorylation and dimerization of Stat1a transcription factor. This is an example of phosphorylation of a transcription factor as an activating step. The IFNg receptor is activated by the binding of its ligand IFNg. This time the receptor is a transmembrane protein located in the plasma membrane. This is the kind of receptor we normally associate with signalling to cells from outside. When IFNg binds to the receptor, the receptor dimerizes, which causes activation of the associated JAK kinase on the cytosolic side of the plasma membrane. JAK kinase phosphorylates the inactive transcription factor Stat1a, thus activating this TF. Phosphorylated Stat1a then able to dimerize with itself, homodimer formation, and the dimer moves into the nucleus where it binds to its response elements in the DNA regulatory region of genes that it controls. You have just learned a signal transduction pathway!
13. 'Unlocking the Gates to Gene Expression' (Fry and Peterson, Science 295:1847(2002). Here I will discuss Panels B and C in this article. Panel A, in which activators recruit remodeling enzymes prior to the formation of the pre-initiation complex (PIC), was discussed in Lecture 17.
Panel B: Here is a new variation on the transcriptional activation theme. At the human IFN-b promoter, upstream activators, shown in green and purple, recruit multiple HAT proteins during assembly of PIC. The initial group of activators bind to a nucleosome-free region upstream of the IFN gene to form an enhanceosome. The HAT called Gcn5p is recruited and acetylates the histones in the nucleosomes in the area of the TATA box. Histone acetylation promotes the binding of SWI/SNF into the initiation site, which disrupts the structure of a promoter-bound nucleosome. The binding of TBP is thus facilitated and the assembly of PIC is completed.
Panel C: In yet another variation, PIC assembly occurs first on the human a1-AT gene promoter, followed by the recruitment of multiple HAT complexes (CBP and P/CAF) and the SWI/SNF complex. This stimulates expression of the a1-AT gene. In this case, the entire upstream region of this gene contains nucleosomes. One activator protein HNF-1 and two general transcription factors, TBP and TFIIB, appear to be bound already to the nucleosome-coated promoter region, even without recruitment of chromatin remodeling enzymes. This prebinding of general TFs allows rapid recruitment of RNA polymerase II and other general TFs. Thus it appears that the entire PIC can be assembled on this promoter in the absence of chromatin remodeling. However, after PIC is assembled, remodeling enzymes are recruited and histone acetylation results in disruption of nucleosomes in the promoter region, and along with the action of a SWI/SNF-like enzyme, transcription is initiated by release of RNA polymerase II from the complex. RNA pol II then moves into its elongation mode, producing a complementary RNA transcript.
